Bottom Line:
We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2.Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I).We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

ABSTRACTDuring anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

Figure 3: stu2-10/mad2Δ cells do not elongate spindles but otherwise progress normally through the cell cycle at 34°C. stu2-10/mad2Δ cells were grown at 25°C, and then elutriated and released at 34°C. (A) Microphotographs taken 135 min after the release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and is shown in blue. (B) FACS® profile shows that cytokinesis happens with the wild-type timing. (C) Sister chromatids separate 90 min after budding. Less than 1% of stu2-10/mad2Δ cells elongate their spindles. Bar, 5 μm.

Mentions:
The metaphase delay of stu2-10 limits analysis of its role in anaphase. Previous studies have shown that malformed spindles are monitored by the spindle checkpoint pathway, a key member of which is MAD2 (Li and Murray 1991; Alexandru et al. 1999). To check whether the stu2-10 arrest was dependent on MAD2, we constructed a double mad2Δ/stu2-10 mutant. We obtained a population of mad2Δ/stu2-10 G1 cells using centrifugal elutriation, released them at 34°C, and monitored cell cycle state by FACS analysis and separation of GFP–CEN dots. Fig. 3 shows that deletion of MAD2 abolishes the stu2-10 arrest in metaphase, and the timing of sister chromatid separation is indistinguishable from wild type. (Compare Fig. 1 C with Fig. 3 C. Use time of the budding as a time reference for cell cycle position.) We therefore conclude that stu2-10 mutant cells arrest in metaphase because they engage the spindle checkpoint. To analyze the role of Stu2 during anaphase, we monitored spindle length in stu2-10/mad2Δ double mutants. Although the double mutant cells progressed through mitosis, no spindle elongation was seen (Fig. 3A and Fig. C). From these results we conclude that Stu2 is required for spindle elongation during anaphase.

Figure 3: stu2-10/mad2Δ cells do not elongate spindles but otherwise progress normally through the cell cycle at 34°C. stu2-10/mad2Δ cells were grown at 25°C, and then elutriated and released at 34°C. (A) Microphotographs taken 135 min after the release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and is shown in blue. (B) FACS® profile shows that cytokinesis happens with the wild-type timing. (C) Sister chromatids separate 90 min after budding. Less than 1% of stu2-10/mad2Δ cells elongate their spindles. Bar, 5 μm.

Mentions:
The metaphase delay of stu2-10 limits analysis of its role in anaphase. Previous studies have shown that malformed spindles are monitored by the spindle checkpoint pathway, a key member of which is MAD2 (Li and Murray 1991; Alexandru et al. 1999). To check whether the stu2-10 arrest was dependent on MAD2, we constructed a double mad2Δ/stu2-10 mutant. We obtained a population of mad2Δ/stu2-10 G1 cells using centrifugal elutriation, released them at 34°C, and monitored cell cycle state by FACS analysis and separation of GFP–CEN dots. Fig. 3 shows that deletion of MAD2 abolishes the stu2-10 arrest in metaphase, and the timing of sister chromatid separation is indistinguishable from wild type. (Compare Fig. 1 C with Fig. 3 C. Use time of the budding as a time reference for cell cycle position.) We therefore conclude that stu2-10 mutant cells arrest in metaphase because they engage the spindle checkpoint. To analyze the role of Stu2 during anaphase, we monitored spindle length in stu2-10/mad2Δ double mutants. Although the double mutant cells progressed through mitosis, no spindle elongation was seen (Fig. 3A and Fig. C). From these results we conclude that Stu2 is required for spindle elongation during anaphase.

Bottom Line:
We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2.Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I).We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

ABSTRACTDuring anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.